LAB 1 WRITE-UP

Initial Machine Testing

The Original Design

The PCR machine, also known as a thermocycler, is a device used make multiple copies of DNA by raising and lowering the temperature of various segments of DNA. Through this process the samples of DNA can be amplified using the polymerase chain reaction to create several copies of the DNA sequence. This technique of raising and lowering temperature to initiate the polymerase chain reaction is crucial to modern medicine as it allows the cloning of vast amounts of DNA to be utilized for other means such as DNA sequencing.

Experimenting With the Connections

The LCD screen produces a output on a screen to show what stages the Opeb PCR is in during the amplification
The Heat block controls the temperature of the Open PCR making it get to high temperature
The heated lid makes the DNA samples not do condensation while the temperature raises in the Open PCR

Test Run

March 5th, 2013 was our first testing the number 12 Open PCR machine.
Downloaded and connected the OpenPCR to a computer with no problems.
Our diagnostic test by running “A Simple Test” worked successfully( The numbers matched very closely from display screen on the Open PCR lid with what appears on the computer.)

Protocols

The Experimental Protocol Planner developed and carried out the methods of a Polymerase Chain Reaction protocol.
Jurisdiction includes: Programming the PCR program, labeling the vials, and interpreting experimental data such as positive and negative controls. Thermal Cycler Program
The program ran from a PCR application for Windows computer was as follows:
Stage One: 1 Cycle, 95 °C for 180 seconds
Stage Two: 35 Cycles, 95 °C for 30 seconds, 57 °C for 30 seconds, 72 °C for 30 seconds
Stage Three: 1 Cycle, 72 °C for 180 seconds
Final Hold: 4 °C
It should look like this:

DNA Sample Set-up
The DNA was taken from two patients and separated into three replicates.
This setup is accurate to how the vials were oriented in the PCR, and are labeled as such:

1.) Use the micro-pipet to dispense 50 μL of PCR reaction mix into 8 vials, be sure to change the transfer pipettes to avoid cross contamination.
2.) The DNA is transferred to its appropriate vial, 50 μL each.
3.) The vial labeled (+) is then given cancer DNA as a positive control for the cancer marker testing and the vial labeled (-) is given no DNA template, then the DNA is dispensed according to the above table.
4.) The vials are ready for the PCR system, which should be programmed according to the Cycler program above.

DNA/ primer mix
The primer is a determinant of the overall outcome of the PCR mix. The primer has distinct characteristics such as:
-18-24 Bases Long
- Balanced distribution of G/C and A/T bases
- Not complementary to each other (5'-3')
This primer acts to binds to the DNA at a specific fragment, one forward and one backwards to read the DNA. <b
The reason there are forward and reverse primers is because the primer binds to a specific 3' end of the DNA, and both single strands of the double helix must be read.
The primer then uses the DNA polymerase to synthesize a the complementary strand; the polymerase only synthesizes DNA with the primer binded to it.

Research and Development

Specific Cancer Marker Detection - The Underlying Technology

Polymerase Chain Reaction, or PCR, is the occurrence in which a segment of DNA is replicated multiple times. For the reaction that we are trying to produce the solution we will be using will contain Template DNA, Primers, Taq Polymerase, Magnesium Chloride and dNTP's.

To begin the process of PCR the solution containing template DNA we take the template DNA and heat up the solution to 95°C for 30 seconds in order to break the hydrogen bonds between the nucleotides. This exposes the bases to allow the primers, which are short sequences of DNA that have been artificially synthesized, to attach to the exposed bases on the template DNA. In this process there are forward primers and reverse primers that attach to the exposed template DNA. After the primers are added, the solution is cooled down to 57°C in order to initiate primer annealing, or binding, to their respective regions. To complete a full strand of DNA the solution must be raised up to 72°C. The raise in temperature activates the Taq polymerase. Taq Polymerase is a protein that allows for binding of dNTP which are individual bases floating around within the solution that act as the subunits that the polymerase use to build new strands. Within the solution the dNTP's bind to the exposed bases starting from the primers. To aid in this process, the added Magnesium Chloride acts as a cofactor to allow polymerase to work and aids with the binding process. Taq polymerase will seek out and react with double stranded parts of DNA and can bind after it attracts individual bases which attach to the primer through a covalent bond, thus creating a new strand of DNA. In a PCR reaction this entire process is repeated multiple times, preferably 30 in order to cancel out other genes and work just for genes that code for cancer.

The reason that the process only codes for the cancerous genes is that the primers were artificially synthesized in order to code for those genes. For the non-cancer gene there will be no binding of the primer since it does not match the same sequence of DNA as the cancer gene. As a result the non-cancerous gene will remain uncoded while the cancerous gene is continuously copied which allows for the development of an new strand by the Taq polymerase and the dNTP's within the solution.